/. Embryol. exp. Morph. Vol. 62, pp. 95-107, 1981
95
Printed in Great Britain © Company of Biologists Limited 1981
Some effects of glucagon on chick embryo
development
By WILLIAM A, ANDERSON 1 AND M. A. GIBSON2
From the Department of Biology, Acadia University, Nova Scotia
SUMMARY
Glucagon concentrations ranging from 116 to 3000/tg/01 ml diluent were injected into
the yolk of chick embryos on incubation days 8,10, and 12. Studies of survival rates, embryo
weights, blood sugars, liver and tibiotarsus glycogen histochemistry, and pancreatic alpha
and beta tissue histogenesis were undertaken during the 9- to 16-day incubation period.
Glucagon dosages of 37-5 and 1500 /*g/01 ml diluent gave the best survival rates. Glucagon
caused an increase in embryo weight, increased liver glycogen storage, a chondrocyte glycogen
storage pattern which correlated with blood sugar levels, an increase in pancreatic beta
tissue and a decrease in pancreatic alpha tissue. Studies of blood sugars following glucagon
treatment showed that most concentrations caused an initial (first 16h) hyperglycemia.
Following this, two general patterns were exhibited: (1) the lower glucagon concentrations
caused hypoglycemia after about 24 h, and (2) the higher concentrations caused a more
prolonged hyperglycemia when administered on incubation day 10 but caused hypoglycemia
when administered on days 8 and 12. Interpretation of these results is based on the contribution of three factors to the expression and duration of the glucagon effect: (1) concentration
of glucagon administered, (2) insulin secretion, and (3) levels of glycogen storage at the
incubation stage of administration.
INTRODUCTION
While a number of studies of the teratogenic influences of insulin on avian
embryogenesis have been reported, fewer studies (referenced below) have been
reported on the effects of glucagon on the developing chick embryo. The
interrelated actions of glucagon and insulin offer the possibility that glucagon
may contribute to the formation of some of the abnormalities which develop
after insulin treatment. This possibility is emphasized by the work of Kalliecharan
& Gibson (1972) on the effects of insulin on the histogenesis of the pancreatic
islets in the chick embryo. They reported a delay in the differentiation of beta
tissue suggesting a delay in the elaboration of endogenous insulin. They also
reported an increased activity of alpha tissue suggesting an increased glucagon
production. The present study was undertaken, therefore, to describe some of
the changes caused by the administration of glucagon to chick embryos.
1
Author's present address: Department of Anatomy, University of Western Ontario,
London, Ontario, Canada.
2
Author's address: Department of Biology, Acadia University, Wolfville, Nova Scotia
Canada BOP 1XO.
4-2
96
W. A. ANDERSON AND M. A. GIBSON
MATERIALS AND METHODS
Fertile White Leghorn chick eggs (Shaver's Starcross 288) were obtained from
a commercial hatchery and incubated at 38 °C for use in this study. The embryos
were studied for body weights, blood sugar levels, histochemically for liver and
chondrocyte glycogen storage, and histologically for amounts of pancreatic
islet tissue.
The eggs were divided into three groups: the untreated series, the glucagontreated series in which the eggs were injected with various concentrations (see
below) of glucagon suspended in diluent, and the control series in which the
eggs were injected with diluent only. Eggs of the latter two series were injected
on one of incubation days 8, 10, or 12, and specimens were collected for study
on each incubation day following treatment until day 16 or until mortality prevented further study. In addition, for the blood sugar study, embryos were
collected at the 2 h, 4 h, 8 h, 12 h, and 16 h stages following glucagon treatment.
The diluent described by Thommes & Firling (1964) was used. It consisted of
2-8 gm lactose, 1-6 ml glycerine, 0-2 ml phenol, and 98-2 ml distilled water. The
glucagon was obtained from Sigma Chemical Company (lot. no. AZ2013),
Orangeburg, New York. The concentrations of glucagon used were 1-16, 4-65,
9-37, 37-5, 150-0 and 300-0 [ig in 0-1 ml of diluent. Treatment consisted of a
single injection into the yolk mass of 0*1 ml of diluent, or of diluent plus
glucagon.
Blood sugar levels were measured by the colorimetric method of Teller (1956)
using Glucostat Reagent (Worthington Biochemical Corporation, Freehold,
New Jersey, U.S.A.). The absorbance was measured with a Spectronic 20
spectrophotometer at a wavelength of 450 [im.
Glycogen storage in the liver and tibiotarsus was visualized histochemically
using tissues fixed in cold Rossman's fluid and prepared with the periodic
acid-Schiff reagent (PAS) method of McManus (Pearse, 1961). Companion
sections were treated with malt diastase to distinguish the glycogen from other
PAS-positive substances. An assessment based on staining intensities was made
of the relative amounts of glycogen stored in the liver. Since all sections were
prepared in an identical manner, the intensity of the staining reaction was
taken as evidence of the amount of glycogen present, and major changes in
staining intensity were assumed to provide evidence of changes in the amounts of
glycogen stored. The staining intensities were calculated from photocytometric
recordings of the light transmitted through the tissue sections before and after
staining.
Pancreas was fixed in Bouin's fluid and prepared with Gomori's chrome alum
hematoxylin-phloxine method (Lillie, 1965) to demonstrate the alpha and beta
cells. The method of Chalkey (1943) was used to determine the relative quantity
of the alpha and beta tissues. An ocular micrometer was moved across the
Some effects ofglucagon on chick embryology
97
Table 1. Survival data (%) for glucagon-treated and diluent-treated
chick embryos
Treatment
Incubation
day of
treatment
8(75)
8 (100)
8 (375)
8 (150)
8 (300)
9 (100)
8(94)
10 (125)
10 (47)
10 (200)
10 (225)
10(419)
10 (108)
12(108)
12 (90)
glucagon/
01
ml)
116
4-65
9-37
37-50
15000
30000
Diluent
116
4-65
9-37
37-50
15000
Diluent
15000
Diluent
Days of incubation
i
9
10
0
17-3
10
200
5 3 0 ". 25-6
68-6' v" 34-6
230
40-3
0
10
83-2
77-8
—
—
—
—
—
—
—
—
•
—
—
—
—
—
—
—
11
12
13
14
15
16
0
0
10-4
15-3
120
0
75-6
57-6
53-2
360
61-7
41-8
951
—
—
0
0
3-4
0-6
5-3
0
73-8
31-2
21
2-5
25-7
20-5
93-7
—
—
0
0
1-8
0
2-3
0
71-3
11-2
0
0
17-7
10-7
92-4
58-3
88-8
0
0
0-5
0
0-6
0
68-8
5-6
0
0
120
3-5
90-9
25-9
870
0
0
0
0
0
0
68-8
1-6
0
0
10-2
2-4
90-9
111
82-8
0
0
0
0
0
0
68-8
0
0
0
0
0
90-9
5-5
78-2
field in units of 100 scale divisions and readings were recorded at every second
stop of the scale. At each reading, the cell (acinar, connective tissue, alpha, and
beta) underlying every 10th scale division was identified and recorded as a 'hit'.
A total of 600 'hits' were recorded for each animal studied, and the amounts of
alpha and beta tissue are expressed as a percentage of this total.
RESULTS
Survival data. Table 1 shows the percent surviving embryos on each incubation
day following treatment. Administration of diluent increased mortality but not
as severely as the administration of glucagon. Administration of glucagon
caused severe mortality within the first 24 h, after which the surviving embryos
showed three general patterns. The lower concentrations, with exceptions on
days 8 and 10, gave high mortalities; the intermediate concentrations gave
lower mortalities; and the highest concentration used (300 jug) gave the highest
mortality.
Embryo weights. The diluent caused a decrease in body weight (Table 2),
necessitating a comparison between diluent-treated and glucagon-treated
embryos to determine the actual glucagon effect. Compared to the diluenttreated embryos, the glucagon-treated embryos showed an increased body
weight. However, in only a few instances did the glucagon-treated embryos
Untreated
Diluent
9-37
37-50
15000
Diluent
9-37
37-50
15000
Diluent
15000
10
10
10
10
12
12
Treatment
(/ig glucagon/
0 1 ml)
8
8
8
8
Incubation
day of
Treatment
1
2
—
—
13-46±l-77(5)
10-77±l-57(5)
1213 ±0-81 (6)
15
11-42 ±1-27 (5)
6-87 ± 0-97 (5)1 10-22 ±0-53 (5)
9-46 ±0-73(5)2 12-55 ± 0-93 (5)1-2 15-31 ±1-32(5)
8-36 ±0-80 (5) 10-72 ±0-90 (5)
9-72 ±1-25 (3)2
9-79 ±0-44 (5)
5-75 ± 0-30 (6)1
6-86 ±1-46 (2)
6-21 ±0-69 (5)
6-86 ± 0-61 (5)2
3-85 ±0-07 (5)1
5-58 ± 0-33 (5)1-2
4-47 ± 006 (5)2
601 ±0-51 (5)1-2
7-30 ±0-27(4)x
9-69 ± 0-96 (3)2
6-85 ± 0-61 (5)2
14
910 + 0-86(20) 9-68 ±1-59 (10)
6-76 + 0-36 (21)1
912 ± 0-38 (5)2
13
7-08 + 0-75(10)
5-22 + 0-45 (5)1
6-85 ± 0-67 (5)2
12
4-42 ±0-33 (10)
3-80 ±0-42 (5)
5-64 ± 0-44 (5)1-2
4-38 ±0-53 (5)
5-35 ± 0-73 (5)1-2
11
Significantly different from untreated embryos (P < 001).
Significantly different from diluent-treated embryos (P < 001).
—
—
315±0-36(13)
2-74 ±0-28 (9)
3 -45 ± 0-37 (5)2
3-22 ±0-49 (5)
3-71 ±0-42 (5)2
2-49 + 0-18(10)
1 -99 + 0-24 (9)1
2-55 ± 010 (5)2
2-40 + 0-45(5)
2-37 + 0-18(5)
—
10
9
Days of incubation
Values are means ± S.D. Number of specimens in parentheses.
Table 2. Weights (g) of untreated, diluent-treated, and glucagon-treated chick embryos on the incubation stages studied
oo
Some effects ofglucagon on chick embryology
99
exhibit body weights which were significantly higher than those of the untreated
embryos. In these instances, the higher concentrations administered at the
later incubation stages had the more prolonged effect.
Blood sugar. The blood sugar levels during the first 16 h following the injection
of glucagon show that most of the glucagon concentrations used caused hyperglycemia (Table 3).
The blood sugar levels measured at 24 h intervals following treatment are
given in Table 4. Most glucagon treatments caused an initial hyperglycemia,
after which the blood sugars showed two general patterns: (1) the lower glucagon
concentrations caused hypoglycemia after about 24 h, and (2) the higher concentrations caused a more prolonged hyperglycemia when administered on day
10 but caused hypoglycemia when administered on days 8 and 12.
Liver glycogen. Liver sections from untreated embryos contained scattered
glycogen granules on incubation days 9 and 10, with sites of accumulation
around the major blood vessels. On incubation days 11 to 15, the staining was
more evenly distributed throughout the lobules. The photocytometric measurements (Table 5) show: (1) a progressive increase in glycogen storage during the
period studied, except for a stage of reduced storage on incubation day 13; and
(2) a major increase in glycogen storage between days 14 and 15.
All glucagon concentrations used, regardless of the day of injection, resulted
in significant increases in glycogen staining (Table 5) for most of the period
studied. On incubation day 15, embryos treated with 1-16 and 150-0//g glucagon
on day 10 exhibited reduced glycogen staining correlating with the hyperglycemic
blood shown by these embryos at this stage.
Tibiotarsus glycogen. In tibiotarsi from untreated embryos on incubation day
9, the chondrocytes of the epiphyses possessed a few glycogen granules. The
early proliferative zone showed little staining, the late proliferative zone stained
more intensely, the early hypertrophic zone gave the most intense reaction, and
the late hypertrophic zone contained little glycogen. The glycogen distribution
on incubation days 10 to 15 was similar and showed a progressive increase in
staining intensity.
In glucagon-treated embryos, changes in the glycogen-staining pattern were
consistently observed only in tibiotarsi from the more severely affected embryos.
These variations, with few exceptions, mirrored changes in the blood sugar levels.
That is, when blood sugar levels increased (e.g. day-9 tibiotarsi treated with
9-37 fig on day 8), there was also increased glycogen storage in the early and late
hypertrophic zones. Conversely, when blood sugar levels decreased (e.g. day-12
tibiotarsi treated with 150 /ig on day 10), both the epiphyses and early hypertrophic zones showed glycogen depletion.
Pancreas. The study of the relative amounts of alpha and beta tissue was
limited to those treatments which had a severe effect on blood sugar levels.
Table 6 shows that the amounts of alpha tissue in the treated embryos were
either similar to or reduced below the values for the untreated pancreas on most
143-24 ±9-27
143-39 ±3-71
15000
Untreated
8
post-treatment
160-00 ±18-89
141-53 ±1-95
154-52 ±11-25
282-67 ±11-97*
147-71 ±1-86
185-63 ±4-57*
19500 ±-5-33*
191-81 ±7-20*
128-27 ±2-37
]Hfours
12
12305 ±11-85
140-64 ±1-95
142-40 ±12-26
214-35± 18-43*
14402 ±1-35
196 06 ±4-47*
188-44±8-87*
257-37 ±5-90*
133-63 ±1-72
* Significantly different from value for untreated embryos (P < 001).
16217±1615
142-97 ±2-92
207-53 ±9-47*
180-83 ±7-22*
14717 ±2-60
116
15000
Untreated
12
12
165-92 ±9-66
261 -32 ±13-26*
150-92 ±2-60
214-78 ±6-67*
132-77 ±1-72
10
10
10
162-20 ±308*
15614±ll-20
238-64±7-58*
129-77 ±300
161-09±8-85*
116
9-37
15000
Untreated
4
2
Treatment
(jig glucagon/
0 1 ml)
Incubation
day of
treatment
Values (mg%) are means ±S.E. of measurements for 10 specimens.
8001 ±802*
145-65 ±310
197-85 ±7-78*
28215 ±13-92*
149-31 ±1-51
153-20±4-51*
163-34±6-17*
181 04 ±608*
131-59 ±2-49
16
Table 3. Blood glucose (mg%) levels for untreated and glucagon-treated embryos measured at 2 and 4 h intervals following
treatment
00 00 00 00
o
o
Diluent
116
4-65
9-37
37-50
15000
Diluent
15000
10
10
10
10
10
10
12
12
Untreated
Diluent
116
4-65
9-37
37-50
15000
Incubation Treatment
day of Og glucagon/
Treatment
01 ml)
144-70±316(38)
15103 ±1718 (5)
81-01 ±15-99 (5)*
39-80 ±3-92 (2)*
159-90 ±7-92 (17)
150-60 ±23-90 (5)
115-85± 10-02 (11)*
14
10
141 -21 ±3-46 (5)
95-40± 10-34 (10)*
64-80±10-59(5)*
49-24 ±7-39 (12)*
290-10±19-37(5)*
186-40± 10-43 (24)*
142-65 ±5-85 (5)
135-40± 16-35 (10)
—
118-00±17-79(5)
139-45 ±24-34 (5)
106-80±ll-18 (26)*
145-15 ±2-31 (5)
150-40±13-96(5) 186-30±4-59 (2)*
129-20±15-39(5) 142-36±9-44 (5) 150-30 ±9-37 (5)
116-70±9-44 (17)* 169-70±4-31 (5)* 171-20±3-36(10)*
134-39 ±6-87 (5)
146-30 ±3-60 (5)
38-28 ±16-67 (5)* 80-86±1308 (5)' 116-20±14-30(5)*
144-67 ±5-20 (5)
11810±2007(6)
Significantly different from untreated embryos (P < 001).
—
—
—
—
31-14±9-27 (2)*
10810±2-93(2)*
65-60±9-28 (2)*
13
58-5O± 11-20 (16)* 11810+ 15-54 (7)* 118-20±28-75(5)
122-23±23-45 (5)
95-50±940 (11)*
127-22±18-11 (4) 1O7-8O± 10-43 (11)* 121-50±42-69 (5)
12
143-76±2-82 (26) 147-35 ±3-36 (17) 150-15±l-74 (48)
141 03 ±4-38 (5)
146-35 ±2-21 (48)
140-24±15-86(5)
11
145-04 ±3-27 (21)
145-55 ±3-78 (18)
10
Days of incubation
Values are means ± S.E. Number of specimens in parentheses.
Table 4. Blood glucose (mg%) levels for untreated, diluent-treated, and glucagon-treated embryos measured at daily intervals following the
stage of treatment
—
8
8
8
10
10
10
10
10
12
Untreated
116
9-37
150-00
116
4-65
9-37
37-50
150-00
15000
Incuba- Treatment
tion
day of (jig glucagon/
treatment
0 1 ml)
9
5-70 ±5-67 (5)
42-90+11-68 (2)*
20-22 + 8-94(4)*
62-20±12-66(3)*
—
—
—
—
—
—
f
11
12
13
24-68 ±9-96 (6)
22-05±702 (6)
13-20±6-13(7)
610±703 (5)
—
—
—
—
20-90 ±6-54 (7)*
71-40 ±12-63(1)* 48-38 ±12-39 (2)*
—
—
68-68±13-52(4)*
—
36-60±10-59(3)*
—
3604 ±9-31 (2)*
—
—
—
6508±10-87 (3)*
—
—
—
79-44+15-48(2)*
—
—
—
4106±10-47(5)* 42-66 ±11-86 (3)*
—
—
38-90±10-12(6)* 41-72 ±10-48 (4)* 56-78 ±11-26 (4)*
—
—
—
56-18 ±9-83 (2)*
* Significantly different from untreated embryos (P < 001).
10
Days of incubation
14
26-63 ±7-98 (3)
—
—
—
—
—
—
52-18 ±10-50 (5)*
54-38 ±11-22 (2)*
63-26±ll-88(3)*
Values are means of 100 readings per specimen ± S.D. Number of specimens in parentheses.
Table 5. Percent staining intensity ofglycogen in liver from untreated and glucagon-treated embryos
75-74 ±10-75 (3)
—
—
—
40-66±505 (2)*
—
—
—
45-66 ±6-44(1)*
74-04±ll-73(3)
15
Some effects of glucagon on chick embryology
103
Table 6 Relative amounts (percentage) of pancreatic islet tissue in
untreated and glucagon-treated chick embryos
Values are means ± S.D. Number of animals in parentheses.
Incubation
day of
treatment
Treatment
(jig glucagon/
01 ml)
Day of
incubation
Alpha tissue
Beta tissue
Untreated
12
13
14
15
4-50±015(3)
6-90 ±212 (4)
8-84±0-47 (3)
10-88 ±3-44 (5)
4-85 ±0-39 (3)
5-50±105(4)
5-40 ±0-53(3)
8-36±3-12(5)
10
37-5
12
13
14
15
4-5O±O-38(3)
4-40±009 (3)2
5-20 ± 1-57 (3)2
1110±6-32(3)
610 ± 0-29 (3)2
7-60 + 0-80 (3)2
5-3O±O-81 (3)
7-6O±117(3)
10
1500
12
13
14
15
410±l-84(3)
3-6O±l-88(3)2
4-80±l-79(3)
10-90±2-53(3)
2-90 ± 0-47 (3)1
3-3O±O-38(3)1
4-70 ±2-36 (3)
1000±2-98 (3)
12
1500
13
14
8-6O±2-36(3)
9-20 ±2-94 (3)
12-20 ± 1-64 (3)1
12-50 ±1-47 (3)1
Significantly different from untreated embryos: 1, P < 001; 2, P < 0 0 5 .
of the stages studied. Two of the three glucagon treatments used (37-5 /*g day
10, and 150-0/Ag day 12) caused increases in the amounts of beta tissue, and one
treatment (150-0 /tg day 10) caused decreased amounts of beta tissue on most of
the stages studied. By incubation day 15, the values for both the alpha and beta
tissues were similar to those of the untreated pancreas.
DISCUSSION
Survival rates. The survival patterns described (Table 1) correlate with changes,
in the blood sugar levels (Tables 3 and 4). The initial high mortality, within the
first 24 h, correlates with the period of hyperglycemia. This hyperglycemia was
most severe following treatment with the 300 /*g concentration and no embryo
receiving this concentration survived the first 24 h. Embryos receiving the other
glucagon concentrations also showed an initial hyperglycemia but this was
reduced within the first 24 h to control or to hypoglycemic levels. It is suggested:
(1) that the initial hyperglycemia together with glucagon's insulinogenic role
stimulates the elaboration and release of insulin, and (2) that this insulin
counteracts the effects of the glucagon and lowers the blood sugars to below
lethal or to hypoglycemic levels. Furthermore, it may be this insulin-induced
104
W. A. ANDERSON AND M. A. GIBSON
hypoglycemia, and not the initial glucagon-induced hyperglycemia, which
contributes to the continuing mortality rate.
Embryo weight. The decrease in body weight caused by the diluent may be
related to its lactose component. Rutter, Krochevsky, Scott & Hansen (1953)
studied the effects of a lactose diet on post-hatched Columbian chicks and reported a reduction in body weight as well as other abnormalities.
Some glucagon treatments caused significant weight increases when compared with the untreated embryos. Thus, the glucagon acted as a growth stimulant, more than simply blocking the depressant effects of the diluent. In contrast,
no significant weight changes following glucagon administration were reported
in rats and rabbits (Root, 1953), White Leghorn chick embryos (Elrick,
Konigsberg & Arai, 1958), or 21- to 28-day broilers (Cavora & Kondra, 1970).
Cavallero (1956), however, reported that glucagon administered to White
Leghorn chick embryos caused significant weight increases.
Liver glycogen. The pattern of liver glycogen storage for the untreated
embryos, including the decrease at day 13, is comparable to those described in
previous reports (Dalton & Hanzal, 1940; Jenkins, 1955; Leibson, Zheludkova,
Plisetskaya & Strabrovsky, 1961a; Daugeras, 1968). While the pancreatic beta
cells show insulin granules at earlier incubation stages, Kalliecharan & Gibson
(1972) demonstrated a major increase in the staining intensity of these cells,
suggesting increased insulin elaboration, beginning on incubation day 13. Also,
Leibson et al. (1961 b) have reported that insulin is present in increased amounts
in the blood plasma on days 11-14. This increased insulin production may contribute to the major increase in liver glycogen storage between days 14 and 15.
Previous studies have reported initial decreases in liver glycogen storage
following glucagon treatment in chick embryos (Grillo, 1961; Thommes &
Firling, 1964; Korec, 1967; and Verne & Hebert, 1968). Grillo studied liver
glycogen during the first 8 h following glucagon injection and reported a
significant decrease during the first 3 h and a return to control values during the
remaining 5 h. In the present study, all liver sampling was undertaken at 24 h
intervals following glucagon injection and missed the short term effects described by Grillo. The present study is, consequently, a continuation of the previous work and has shown that the initial glycogen loss is followed by a more
prolonged period of increased storage. Pincus & Snedecor (1956) stated that
glucagon's hyperglycemic effect resulted from its glycogenolytic activity. Thus,
liver glycogenolysis accounts for the initial glycogen loss from the liver contributing to the hyperglycemic blood. The important suggestion here is that this
initial reaction appears to be counteracted by the secretion of insulin. Thus, the
initial hyperglycemia is reduced to untreated or to hypoglycemic levels, and the
liver glycogen is increased to a storage pattern above that of the untreated
embryos. Ui, Claus, Exton & Park (1973) have shown that glucagon stimulates
the transamination of glutamate to L-ketoglutarate and also increases phosphoenolypyruvate activity. Thus, the protein reserves in the yolk sac may be
Some effects of glucagon on chick embyrology
105
mobilized by glucagon in the form of glucose, and this gluconeogenic effect of
glucagon, as well as its insulinogenic effect, may contribute to the concurrent
appearance of hyperglycemia and increased liver glycogen staining.
Blood sugars. The blood sugar levels of the untreated embryos reported in this
study are in general agreement with those reported by Arsenault, Gibson &
Mader (1975) and Zwilling (1948).
Most of the glucagon treatments caused an initial hyperglycemia. This has
also been reported by Thommes & Firling (1964). Following this initial hyperglycemia, the blood sugar studies illustrate the contribution of three factors to
the expression and duration of the glucagon effect. (1) The concentration of the
glucagon administered influenced the blood sugar levels. For example, 24 h
after the day-10 injections, the lower glucagon concentrations caused hypoglycemia and the higher concentrations caused hyperglycemia. The latter
observation indicates that sufficient glycogen reserves were present for mobilization. The lower concentrations, therefore, were not sufficiently concentrated to
mobilize these reserves. (2) The secretion of insulin also influenced the expression
of the glucagon effect. Leibson et al. (1976) showed that insulin is released before
incubation day 12 and Table 6 shows that the beta tissue is often increased in
amount by the administration of glucagon. Thus, it is suggested that the initial
hyperglycemia is reduced by the release of additional insulin and that it is the
increased insulin that maintains the hypoglycemia. (3) The time of treatment,
and the glycogen reserves available at that time, influenced the glucagon effect.
This is illustrated by the observation that the same concentration administered
on different incubation days elicited varying responses. For example, the 150 /ig
treatment caused hypoglycemia after 24 h when administered on days 8 and 12
but a more prolonged hyperglycemia when administered on day 10. The liver and
yolk sac are two important sites of glycogen storage. Studies of untreated liver
tissue (Table 5) showed a low level of glycogen storage on incubation days 9 and
13, and a high level on days 11 and 12. Juurlink & Gibson (1973) demonstrated a
weak glycogen reaction in the yolk sac on day 13.
Tibiotarsus -glycogen. The glycogen staining pattern described for the
untreated tibiotarsus is similar to that reported by Ho & Gibson (1972) and
Rabinovitch & Gibson (1972). In the glucagon-treated embryos, only the more
severely affected tibiotarsi showed variations from the untreated glycogen
pattern and these variations usually mirrored the changes in the blood sugar
levels.
Pancreatic islets. In untreated embryos, during the 12- to 15-day incubation
period, the pattern of increasing amounts of alpha and beta tissues is similar to
that described by Kalliecharan & Gibson (1972) and Arsenault & Gibson (1974).
In glucagon-treated embryos, the changes in the amounts of alpha and beta
tissue showed no consistent variation which could be correlated with hypoglycemic or hyperglycemic blood. However, many of the embryos studied did
show a reduction in the amount of alpha tissue and an increase in the amount of
106
W. A. ANDERSON AND M. A. GIBSON
beta tissue. The major exception to this pattern was the reduction in beta tissue
shown by those embryos treated with 150 /*g of glucagon on day 10 (Table 6).
These were the embryos in which the blood returned to hyperglycemic levels
(Table 4). Peterson & Hellman (1963) reported increased beta tissue and decreased A2 cells in older rats following prolonged glucagon treatment.
It is suggested: (1) that the exogenous glucagon, which has an insulinogenic
role (Crockford, Porte, Wood & Williams, 1966; Devrum & Recant, 1966; and
Samols, Marn & Marks, 1966), caused an increased development of beta tissue,
and (2) that it reduced the release of endogenous glucagon and delayed the
histogenesis of the alpha tissue.
One reason for the present study was to examine the possibility that increased
glucagon release might contribute to those abnormal patterns of development
that appear following the administration of insulin. Rumplessness, beak
deformities, micromelia, etc. did not develop after the glucagon treatment.
Abnormalities in skeletal histogenesis (Rabinovitch & Gibson, 1972; and others)
were not observed in the glucagon-treated embryos. Insulin treatment causes
prolonged hypoglycemia (Zwilling, 1948; Arsenault et ah 1975); whereas,
glucagon caused an initial hyperglycemia followed by hypoglycemia. Insulin
treatment causes a delay in the histogenesis of the beta islets and increased
activity of the alpha islets (Kalliecharan & Gibson, 1972); whereas, glucagon
treatment frequently led to the opposite effect. The present study suggests,
therefore, that exogenous glucagon and insulin-stimulated endogenous glucagon
may have somewhat different roles. Exogenous glucagon stimulates insulin
secretion and it is the increased insulin that contributes to many of the abnormal
patterns described in this paper. Endogenous glucagon, stimulated as the result
of insulin treatment, possibly functions more to counteract the effects of the
exogenous insulin, and does not appear to contribute to those gross morphological abnormalities described as the insulin syndrome.
This work was supported by a grant from the Natural Sciences and Engineering Research
Council of Canada.
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(Received 28 August 1979, revised 2 October 1980)